STIMULATION AND SUBSTITUTION OF BONE ALLOGRAFT AROUND NON- CEMENTED IMPLANTS

Transcription

1 STIMULATION AND SUBSTITUTION OF BONE ALLOGRAFT AROUND NON- CEMENTED IMPLANTS Ph.D. thesis Thomas Bo Jensen Faculty of Health Sciences University of Aarhus 2003 Side 1 af 40

2 Abbreviations...4 Abstract...6 Introduction...7 Background...8 Grafting in reconstructive surgery of the hip...8 Biomechanics of impacted morselized bone allograft...9 Incorporation of impacted bone allografts...10 Risks and disadvantages associated with the use of bone graft...9 Bone graft substitutes based on osteoconductivity...15 Bone growth factors and implant healing...12 Enhancement of bone grafts and osteoconductive bone substitutes...15 Materials and methodological conciderations...17 Ethical conciderations...17 Animals...17 Observation time...17 Implants...17 Experimental model...17 Design...18 Grafting materials...18 Mechanical evaluation...20 Histomorphometry...21 Reproducibility...21 Statistics...22 Results...22 Exclusions...22 Study II...23 Study III...25 Study V...26 Discussion...27 Side 2 af 40

3 This thesis is based on the following papers and manuscripts: I: Jensen-TB, Overgaard-S, Rahbek-O, Lind-M, Bünger-C, Søballe-K. Osteogenic Protein-1 Device Increases Bone Graft Resorption and Bone Formation Around Cementless Implants. Acta Scandinavica Orthopaedica,2002, 73(1) p31-39 II: Jensen-TB, Overgaard-S, Rahbek-O, Lind-M, Bünger-C, Søballe-K. Osteogenic Protein-1 Device Increases Fixation of Implants Grafted with Morsellized Bone Allgraft and ProOsteon. An experimental study in dogs. Resubmitted J. Bone Joint Surg. (Br), 2002 III: Jensen-TB, Overgaard-S, Rahbek-O, Lind-M, Bünger-C, Søballe-K. The influence of OP-1 d Device on Mechanical Properties of Impacted Morsellized Bone Allograft after 3 Weeks. Manuscript 2003 IV: Jensen-TB, Overgaard-S, Rahbek-O, Søballe-K, The Influence of Platelet Rich Plasma on Osteointegration of Fresh Frozen and Processed Bone Allograft. Submitted J. Arthro V: Jensen-TB, Overgaard-S, Rahbek-O, Søballe-K, Does Platelet Rich Plasma Influence Fixation of Non-cemented Implants. Resubmitted J. Orthop. Res Side 3 af 40

4 Abbreviations AGF: Autologous growth factor BMP: Bone morphogenic protein EGF: Epidermal growth factor FGF: Fibroblast growth factor HA: Hydroxyapatite IGF: Insulinlike growth factor OP-1: Osteogenic protein-1 PC: Platelet concentrate PDGF: Platelet derived growth factor PRP: Platelet rich plasma TCP: Tricalciumphosphate THA: Total hip arthroplasties Ti-6Al-4V: Titanium-6 aluminium-4 vanadium Definitions Bioactive materials: Materials which elicit or modulate biological activity Biocompatible: The ability of a material to perform with an appropriate host in a specific application Cytokine: Poly peptide regulator of cell-tocell interaction in relation to the immunological system Graft: -autograft:bone tissue harvested from and implanted in the same individual -allograft: Bone tissue harvested from one individual and implanted in another individual, same species -Xenograf: Harvested from one species and implanted in another Growth factors: Polypetides which act as signalling agents for cells Histomorphometry: Quantitative evaluation of tissue Implant: A device made of a biomaterial that is intentionally placed in the body covered totally or partly by an epithelial surface Implant in- or ongrowth: Bone corverage of the implant surface in percentage Bone incorporation: Integration of a biomaterial by bone Osteoconduction: A process where tissue involved in bone formation is lead conducted on the surface of the biomaterial Osteogenesis: Local bone formation occuring when bone forming cells are transplanted from one site to another Osteoinduction: Biochemical stimaulation of new bone formation at an ectopical place Resorption: Reduction of a material due to dissolution or cellular activity Shear: Force or stress occurring under displacement of two parallel surfaces Strain: Relative deformation of an object Stress: The forces that develop within a material when external load is applied Side 4 af 40

5 Preface This thesis is based on experimental studies performed at Orthopaedic Research Group, Department of Orthopaedics, Aarhus University Hospital during my employment as a Diploma student in 1997 (grant from Aarhus University) and during my enrolement as a research fellow in at the Department of Orthopaedics, Amtssygehuset, Aarhus University Hospital (grants from Danish Rheumatism Assiciation and Aarhus University). All surgical procedures and animal handling was done at the facilities of the Institute of Experimental Clinical Research. Preparation and sectioning of tissue and following evaluation was done at Orthopaedic Research Lab. My supervisors were professor Kjeld Søballe, M.D., D.M.SC., professor Søren Overgaard, M.D., D.M.SC. and professor Cody Bünger, M.D., D.M.SC. who took valuable time of to solve problems and kindly commented my work. A special thank to Søren Overgaard for support during the operationspending weekends in the operation room.. I owe speciala sincere gratitude is expressed toto Kjeld Søballe for his astonishing enthusiasm and his involving me in studies and arranging my stay at theintroducing me to Orthopaedic Biomechanics Laboratory, Hennepin County Medical Center, Minneapolis, MN, US in A special thank is directed to Joan Bechttold, Ph.D. for comments to the manuscripts. Professor Fleming Melsen, M.D., D.M.Sc. is thanked for is friendly attitude and his expertise in the evaluation of the bone-implant specimen. My co-workers Ole Rahbek, M.D., Ph.D. and Martin Lind, M.D., Ph.D., D.M.Sc. are thanked for good assistance and comments to the articles. These studies could not have been done without the knowledge and skills of lab technicians Anette Milton and Jane Pauli. Acknowledgements The studies were financially supported by The Danish Rheumatism Association, the Danish Medical Research Council, Institute of Experimental Clinical Research, the Velux Foundation, Beckett foundation and the Aarhus University. The following companies contributed with materials: Biomet: Implants Stryker Biotech: OP-1 device Interpore-Redcross: ProOsteon 200 Side 5 af 40

6 Abstract Bone grafting is important in the revision of failed total joint replacements. However the clinical results on revisions still do not match those of the primary inserted arthroplasties. Histological examinations of impacted bone in revision surgery often show incomplete incorporation and significant subsidence of the prosthesis is often seen. One aim of the thesis was to investigate if bone growth factor osteogenic protein-1 (OP-1) or platelet rich plasma (PRP) increases fixation and bone incorporation of bone allografted implants. Bone allograft, especially fresh frozen, is associated with a number of potential disadvantages and risks however. Risks of viral transmission, bacterial contamination and insufficient supply might limit the use of bone allograft. Different processing techniques almost eliminate the risk of viral transmission or bacterial contamination. Substitutes such as granular ceramics might replace or expand the volume of bone allograft in the future. The thesis is based on five experimental in vivo studies in canines. In all studies, we used nonloaded, stable, hydroxyapatite (HA) coated implants surrounded by a gap. Observation time was three weeks. Evaluation was based on histomorphometry and mechanical push-out tests. In study I and II we examined the effect of recombinant human OP-1 in combination with bone allograft, HA granules or a composite of those. We showed, that OP-1 not only stimulates bone formation but also accelerated bone graft resorption dramatically. As a consequence, OP-1 did not increase fixation of bone allografted implants. Since ProOsteon is very slowly resorbed, OP-1 enhanced mechanical stability of implants grafted with HA granules. In study III we investigated the influence of OP-1 on the mechanical properties of bone allograft and HA-granules after three weeks. OP-1 increased all mechanical parameters of bone allograft and HA-granules. In study IV and V we focused on platelet rich plasma (PRP) as a source of growth factor. We found no effect of PRP alone or in combination with bone allograft. We found no influence of processing by defatting, irradiation and freeze drying on the incorporation of impacted bone allograft. Side 6 af 40

7 Introduction An increasing number of THA is done annually 81. The 10 year survival is now reported up to 97 % and mean age at the primary replacement is 68 years 49. Therefore the majority of patients receiving their first replacement at an old age will never need a revision. Survival of an arthroplasty depends on age, gender and diagnosis and with a three years survival of 94% and 10 year survival down to 80% among men younger than 55 years, the number of revisions are expected to increase in the future 49,81. Aseptic loosening is estimated to be the cause of failure in 79% to 89% of all cases in Scandinavian countries 49,115. In general, revision THA have poorer clinical results, higher costs, longer recreation time and shorter longevity 92,115. One major problem in revisions is periprosthetic osteolysis and the success of revision arthroplasty is highly depended on restoration of the bone stock and creation of a stable implant. The use of bone allograft (morsellized or/and structural) has become routine for many surgeons. Under optimal conditions the impacted morsellized bone graft provides initial mechanical support and creates a newly formed living bone stock which support the prosthesis and might make future re-revisions easier. In vitro studies show, that revision of the femoral component using morsellised bone graft followed by cementing with a collarless prosthesis with a polished tapered stem restores the integrity of the proximal femur and provides immediate stability of the implant 83. However histological analysis of retrieved impacted bone allograft often show incomplete bone incorporation 17,79,93,161. Follow up on cemented prosthesis inserted with impaction have not been conclusive. Reports from the innovators have shown good short- and middle term results at a level similar to primary arthroplasties 80. However high incidence of subsidence especially after massive loss of bone stock has been reported in later studies 35,36,41,91,160 (table I). A large number of bone growth factors has now been isolated and studied in vitro and in vivo. Among the BMP s, most focus has been put on BMP-2 and -7 which are now available for clinical use. The beneficial effect on fracture healing has now been documented in humans 39, but the effect on grafted and non-grafted implants is still unclear. PRP is a concentrate óf platelets in plasma containing a cascade of different growth factors and other cytokines capable of bone stimulation. By mixing bone allograft with purified growth factors or PRP bone invasion might be increased 88 and implant fixation and thereby possibly long term result could be improved. Fresh frozen bone allograft for impaction is preferred in Denmark and other countries. The use of fresh frozen bone allograft is associated with possible immunological reactions, infections and limited supply however. Several processing techniques such as defatting, irradiation, treatment with antibiotics and freeze-drying have been suggested to decrease the potential disadvantages associated with the use of fresh frozen bone allograf 26. Bone substitutes are widely used in orthopaedic surgery, however no substitute has so far been used in larger scale to replace bone allograft in revision of failed THA 100. Ceramics such as HA or TCP might substitute bone allograft. However since the bone stimulating effect is mainly osteoconductive, a combination between a ceramic and a growth factor might be necessary to replace bone graft. Aim (bliver udvidet) The aims of this Ph.D. thesis is to answer the following questions: -Can ProOsteon substitute or extend the volume of fresh frozen bone allograft? -Is there any positive or negative effect of adding growth factor OP-1 to bone allograft or ProOsteon? Side 7 af 40

8 -Does PRP increase fixation and incorporation of implants grafted or non-grafted? -How does processing of bone allograft effect implant fixation and bone incorporation?was to test the following hypothesis: -In study I and II we tested the hypothesis that HA granules or a mixture of HA granules and bone graft can replace bone graft alone. Also we hypothesised, that the addition of OP-1 to the grafting materials increased the bioactivity of the graft and thereby increased stability of the implant. -In study III we tested the hypothesis that OP-1 increases the mechanical properties of impacted HA granules whereas it impairs the mechanical properties of bone allograft due to accelaretd bone graft resorption. -In study IV we tested the hypothesis, that processing by defatting, freeze drying and irradiation impairs the bioactivity of fresh frozen bone allograft. Also we tested the hypothesis that PRP increases bioactivity of processed and fresh frozen bone allograft. -In study V we tested the the hypothesis that PRP increases implant bone incorporation and gap healing alone or in combination with bone allograft. Background Grafting in reconstructive hip surgery Aseptic loosening of THA is often associated with losed bone stock on the femoral and acetabular site. Four different strategies can be used to obtain stability of the revised femoral stem. 1) A short stem designed to fill out the proximal osteolytic defect with proximal fixations is one possibility with minor bone loss. 2) Another approach is to use a long cementless stem with distal fixation leaving an empty proximal defect. This empty proximal defect may be left ungrafted. 3) Simple recementation with a long stem is a third possibility especially in older patients 92. 4) A last possibility is reconstruction by impacted bone allograft around cemented or cementless stems. Restoration of the bone defects by bone grafting may make future revisions easier. Therefore it is recommended to younger patients and to patients with major bone deficiencies. Morsellized impacted bone grafting for cemented revision was introduced by Sloof in 1984 on the acetabular site based on Hastings and Parkes operation technique on protrusio acetabuli using autograft and cement 132. On the femoral site, the use of impacted morselized bone allograft was first introduced without cement in Exeter in Due to high subsidence, cemented technique was introduced in 1987 and subsequent development of operation technique and instrumentation was done as an cooperation between Exeter and Nijmegen. The first publications based on short term results were excellent. However, unpredictable early subsidence of the stem was later reported 35,91. At the beginning, impaction bone grafting mainly included less severe bone loss 41, whereas later studies included more severe bone defects 110,160 (table I). In one study, subsidence was found in 38% of the patients with an average subsidence of 10.1 mm 91. High subsidence has been associated increased risk of loosening of primary inserted prosthesis 59 and also with pain 60. Recent studies have on revision THA have shown similar results when comparing cemented and cementless Exeter X-change technique 110. Side 8 af 40

9 Subsidence and re-revision rates are dependent on multiple factors of which the individual surgeons experience and skills, prosthesis design and the size of the femoral defect seems to be the most important predictors 94,110,160,162. Allograft is more often used in revision surgery than autograft due to required high volume of graft. Cortical or corticocancellous struts harvested from cadavers are mostly used to reconstruct uncontained bone defects. Special attention has been put on cortical struts to strengthen the cortical shell prior to impaction or cementation. Table I: Results of femoral revisions using cemented impaction technique and collared stems Author Number hips Patients Gie, Grade I: 9% Grade II: 48% Grade III: 43 Grade IV: 0 Elting Grade I 34% Grade II 40% Grade III 21% Grade IV 5% Bone defect a Follow up Number of rerevisions months Subsidence 2 20% >5 mm 4%>10 mm 2 years 2 48 % subsided Eldridge No data 1 year 0 11% >10 mm Melding No data 30 months 2 35% 4-31 mm Van Biezen Grade III: 43% Grade IV:57% a: bonedefects quantified according to Endo-Klinik months 0 5% >10 mm Risks and disadvantages associated with the use of bone graft The use of bone allograft does carry potential risks of transmitting virus such as HIV, HBV and HCV 2,10,86,95,124. Even though the load of HIV in bone is low, experimental studies and clinical cases show, that HIV transmission is possible 10,16,86,95,124. One clinical case has been reported where bone graft was harvested from a cadaver in the U S. Fifty-three persons received bone grafts of whom four persons received fresh frozen bone allograft. Three of four patients receiving fresh frozen allograft were with HIV whereas none of the patients receiving freeze dried bone allograft were transmitted 124. Different processing techniques decreases the risk of transmitting virus. Ethanol destroys HIV. However, there is concerns if ethanol penetrates cortical bone sufficiently. Bone marrow has a heavy load of HIV which is decreased by defatting and lavage 10. Todays practise is screening the donor for hepatitis and HIV and no cases of HIV transmission have so far been reported using graft from tested donors. Bone grafts also carry the risk of bacterial contamination. Even though cultures taken at the time of harvesting are negative, they might occasionally be positive when it is later thawed and result in Side 9 af 40

10 deep infection 2,141,146. Again, processing of the allograft by soaking in antibiotics, ethanol or irradiation decreases the risk of contamination. However, even though various methods of preserving, processing and sterilization might decrease the risk of transmission, the most important prevention is sufficient medical and social history of the donor and donor screening. Even though certain donors are excluded, the mineral density of the donated femorla head varies with the general bone quality and hence the quality of banked bone allograft is not consistent 47,109. The demand of bone allograft depends on the number of revisions and also the number of femoral heads needed in each operation. Both parameters are expected to rise 40 and the supply of femoral heads might not be sufficient in the future. Consequently alternative operation techniques to impaction such as distal fixation or substitutes to bone allograft might be used in the future. Biomechanics of impacted morselized bone allograft The biomechanical properties of morsellized bone allograft alone have been investigated in different experimental set-ups 14,42,155. Impacted morselized bone allograft has the mechanical characteristics of an elasto-viscoplastic material 42. The impacted bone recoils immediately after impaction which explains the relatively high initial stability the prosthesis. Stability of a grafted implant is dependent on a number of parameters such as impaction pressure, chips size and bonegraft properties and type of prostheses 153,155. Freeze-drying of trabecular bone increases strength and stiffness, however rehydration makes freeze dried bone similar to fresh frozen bone allograft 19. The combination of freezedrying and irradiation seems to weaken the trabecular bone 24 and freeze drying prior to irradiation is worse than the irradiation prior to freezedrying 114. Bigger size and partly defatted bone chips increase stability of cemented cups 153. Usually bone chips with diameters from 3-5 mm are recommended. Also higher impaction force increases initial fixation however cautions should be taken to prevent fractures 91. In cadavers, the initial stability of cemented stems inserted with impacted bone allograft is lower than cemented stems inserted without impacted graft but better than uncemented stems with no graft 11,83. The biomechanical properties of impacted bone graft changes when tissue invade the graft. Fibrous ingrowth alone increases strength 148. The biomechanical properties of bone chips impacted into rabbit condyles were similar to the original bone after three weeks 173. However the bone ingrowth was better than would be expected in bone allograft in humans. Incorporation of impacted bone allografts Lamerigt described the events of bone incorporation of impacted bone allograft as invasion of the bone graft by a front of vascular fibrous tissue after which osteoclasts resorbed the dead bone graft, followed by woven bone apposition on the graft remnants 70. This front does not go all the way from the periphery to the cement or implant surface. Usually three layers in the grafted area around cemented implants can be identified histologically: an inner zone with fibrous tissue and pieces of allograft chips, a middle zone with bone formation and neocortex and an outer zone consisting of cortex 93. Bone incorporation of impacted bone allograft is fastest during the first six months 79 and radiographic examinations show little change after two years 36. Specimens of impacted graft have been taken as biopsies or autopsis in humans around cemented acetabular and femoral components (table II). Usually a higher degree of bone ingrowth into the graft is seen on the acetabular side compared to the femoral site. Incorporation of impacted morselized bone allograft has also been tested in various animal models. However since bone turnover is higher in the usually young animals and because the anatomy often allows only smaller gaps, bone incorporation and graft remodelling are often reported to be greater than the findings from human studies 73,126,173. Side 10 af 40

11 Many factors influence the rate and extent of bone graft incorporation including immunological response, processing of the graft, size of the bone chips and porosity of the impacted graft particles, thickness and tightness of the graft, load and stability and the bone forming capacity of the recipient. Table II: Histological findings in incorporated impacted bone allograft in humans around implants Implant Cemented femur Component Cemented femoral component Cemented femoral component Acetabular Component Acetabular component Source of specimen Number of specimen Observation time Autopsy 1 6 months Fibrous invasion in the proximal part Biopsies months Biopsies and autopsies months Biopsies months Biopsies months Ingrowth Graft Author Fibrous tissue in the innerzone, new bone in the periphery Usually fibrous invasion Fibrous tissue dominated in the innerzone, bone in the periphery Graft mostly embedded in fibrous tissue Bone chips embedded in fibrous tissue Still chips after 8 years Bone chips remodelled in the periphery, few chips in the innerzone Nonincorporated graft in some areas regardless of time Ullmark 154 Nelissen 93 Linder 79 Buma 17 Van der Donk 161 Allografts are rarely tested for histocompalibility eventhough frozen bone allograft contains cell debris with antigens. Studies have shown, that recipients are being sensitised towards the HLA-type of the graft 144. Mismatched fresh bone allografts are poorly integrated 56,144 and host immune response might play a role in incorporation 43,44. One approach to impair the rejection could be systemic treatment with immunosupressive drugs used for transplantation of organs 43,44. However taken the side effects into account, that is not clinical relevant. Another approach to decrease the immunological response is to decrease the load of antigens. Freezing alone decreases the immunological response 144. Removing the bone marrow cells by lavage and defatting improve bone incorporation 9,56,151 probably because the load of cells is decreased. Removal of cartilage from the femoral head is of great importance of bone incorporation 161. The impact of load on incorporation of bone graft has been proved in a number of experimental studies 163. Wang showed in a rabbit tibia that a loaded stem increased graft resorption and bone formation compared to an unloaded stem 167. Moreover Donk S found a bigger area of active bone incorporation in loaded impacted bone compared to unloaded 163. Since bone around a femoral component is not equally loaded, the histological findings of graft around prostheses might differ according to the anatomical location with more graft replaced by bone distally compared to more proximal 156. Side 11 af 40

12 The speed of bone formation and the mechanical properties during remodelling differs between cortical and trabecular bone due to differences in porosity. Massive cortical grafts are gradually resorbed prior to invasion of vessels and might only be partly substituted by bone 37,46. The fact that cortical graft is resorbed prior to new bone formation might lead to mechanical failure even though it is initially mechanical sufficient 37. On the contrary, cancellous bone allograft serves as a scaffold, where bone is formed on the surface of the trabecular of the bone graft. For that reason, mechanical strength of cancellous graft tends to increase during invasion of bone. The bone allograft is gradually resorped during remodelling of the construct. By soaking the graft in a solution of bisphosphonates, resorption of the graft can be postphoned 7. Porosity is decreased by impaction of the morsellized bone allograft, compared to cancellous bone 147 delaying bone incorporation. Whether the mechanical properties of impacted bone allograft during remodelling is similar to cortical or cancelleous bone is not well known. The importance of BMP s in non-demineralized bone allograft is still not clear. Processing of bone allograft chemically, by irradiation or heat might inactivate BMP s and other growth factors contained in the bone matrix. The growth factors can be demasked by demineralising the bone (DMB). Such bone graft is commercial available and has been proven to be osteoinductive and will usually be incorporated with bone faster than non-demineralised allograft. Application of DMB around implants has so far not been encouraging 22. Bone graft substitutes A number of bone graft substitutes has been suggested to replace morsellized bone allograft around implants due to the disadvantages associated with bone allograft described above. Although e.g. growth factors and bone marrow aspirates improves gap healing, they do not provide initial mechanical support and can therefore not replace bone allograft in revisions. Neither can DBM due to the poor mechanical properties. Mechanical properties of ceramics like HA and TCP granules have been tested 12,152,164,173. They do not have the visco-elastic properties as previously described for bone allograft. Therefore they will not recoil and the initial fixation of implants surrounded by only HA or TCP granules might not be sufficient 164. However stability of cemented femoral prostheses grafted with porous HA/TCP composite and bone allograft have shown promissing results 12,164. Table III: Animal studies on bone graft and bone graft substitutes around non-cemented implants Graft Model Observation Animal Resultat Author periode Autograft THA-revisions, fiber-ti implants 12 weeks Canine +++ McDonald 9 Allograft Revision THA, fiber Ti implants 12 weeks Canine +++ McDonald 9 Autograft Revision THA, fiber Ti implants 6 months Canine +++ Turner 152 Autograft Revision THA, fiber Ti implants 6 months Canine +++ Turner 152 HA/TCP Revision THA, fiber metal Ti 6 months Canine 0 Turner 152 granules implants Allograft 2 mm gap model, Ti implants 6 weeks Canine +++ Soballe, K 139 Allograft 2 mm gap model, HA-coatede 6 weeks Canine +++ Soballe, K implants 139 Allograft THA, HA-coated implants 6 og 12 weeks. Goat ++ Schreurs,B HAgranules Allograft/ HA 3 mm gap model, HA-coated 3 weeks Canine 0 Study I implants 3 mm gap, HA-coated implants 3 weeks Canine ++ Study II 0 0 W 126 Side 12 af 40

13 granulae Allograft 2.5 mm gap model, HA-coated implants 3 weeks Canine +++ Study IV Ceramics have not shown same level of bioactivity as bone allograft 27,152 but one recent study has indicated, that ceramics might be used as an extender of bone allograft around femoral stems 111. Resorption of ceramics depends on structure and chemical compound. TCP is resorbed faster than HA 15,31,129. Due to the differences in mechanical properties between ceramics and bone graft and since some HA granules might never resorp, bone incorporated HA granules might never obtain the same mechanical characteristics as bone alone 173. It is not known if implants surrounded by such composite have inferior survival. Also Bioglass (45S5) has been suggested to replace bone allograft. Bioglass is incorporated at a faster speed compared to HA granules and is faster resorbed 99,101. Bioglass has recently shown promising results as an extender of bone allograft around revision hip prosthesis 34. Bone growth factors Bone growth factors are polypeptides secreted by bone- and other cells (table IV) providing a mechanism for altering cell behaviour such as division, differentiation and matrix synthesis. The exploration and identification of bone growth factors have taken more than a century. In 1889 Nicholas Senn used bovine demineralized bone matrix to fill defects after osteomyelitis and found, that new bone developed. Huggins discovered in 1931, that epithelia from bladder and urether resulted in ectopic bone formation when it was implanted in fascies of guinea pigs. A mile stoe was the article in 1965 by Urist discovering that DBM induced ectopic bone bone formation and later he isolated BMP s which had greater bone inducing effect than DBM BMP s were initially isolated from bovine bone until 1988 when Wosney produced recombinant human BMP Bone growth factors have a number of potential surgical applicationsmight be used in the future in a number of clinical situations including treatment of pseudoarthrosis secondary to impaired bone healing, spinal fusions and bone reconstruction after loosened implants and tumor resection. Table IV: Examples of growth factor sources Growth factor TGF-β BMP FGF PDGF Source Platelets, leucocytes, osteoblasts, chondrocyttes Chondrocyttes, osteoblasts, urether- and bladderepithel Monocyttes, macrophages, osteoblasts, chondrocyttes Platelets, monocyttes, macrophaghes At the moment bone growth factors BMP-2 and BMP-7 are commercially available. OP-1 (BMP-7) is approved in the treatment of nonunion of the tibia of at least 9 month duration, secondary to trauma in skeletally mature patients, in cases where previous treatment with autograft has failed or use of autograft is infeasible 39. Also DBM and kits for preparing PRP are commercialiced. Platelet concentrates Fracture repair is regulated by a number of systemic and local factors. In all stages of fracture repair, growth factors, cytokines and other proteins produced by platelets, leucocytes, and macrophages are believed to play an important role 88,143. A haematoma is formed at the site of a fracture; platelets are activated by collagen exposure leading to fibrin clotting and platelet Side 13 af 40

14 aggregation. Thrombin initiates platelet degranulation. A large number of growth factors such as platelet derived growth factors (PDGF), transforming growth factor-β (TGF-β1), insulin-like growth factors (IGF) and epidermal growth factor (EGF) have been isolated from platelet α- granules 71,71,71,131,131,131,169. Other factors such as β-thromboglobulin and macrophage inflammatory protein-1 α, which are mediators of inflammation with various effects on white cells including macrophages are also found in platelet granules 65. Platelet concentrates are usually used in a fraction of autologous plasma. Marx introduced the name platelet rich plasma. Later a number of different companies has patented kits for PRP preparation and call the final product AGF, Symphony, PCCS etc. PRP is basically based on the idea that more is better. Local application of PRP might increase the local concentration of growth factors and thereby stimulate healing of damaged tissue such as bone defects or ulcers 66,67,84. Since platelets are active in fracture and tissue repair, a concentrate might be beneficial. Furthermore PC stimulates osteoblast proliferation in vitro 131,131,131. Local application of platelet concentrates (PC) has been suggested to enhance healing of damaged tissue such as bone defects or ulcers{24}{464}{465}. Experimentally, platelet concentrate increased bone ingrowth in bovine cancellous bone in a rabbit calvarial defect model 62. Also Kim et al found more bone in contact with dental implants in the group treated with DBM and PRP than DBM alone after 6 weeks but not after 12 weeks 63. Jensen et al found effect of AGF, a platelet concentrate processed with the use of a commercialized filter, in combination with allograft around non-ha coated implants in canines 54. However they found no effect of AGF with no bone graft around loaded implants 54 indicating, that platelet concentrates need to be mixed with a material to keep it on location preventing it from dilution. Platelet concentrates ia number of articles describes the use of platelet rich plasma to enhance bone regeneration and healing of soft tissue in humans 4,66,142,165,170 but only trial using PRP to enhance bone healing has been published 88. In a prospective randomized clinical study, 88 patients with mandibular defects were randomly treated with morselized autograft alone or autograft+prp. PRP was increased by 238% compared to base line. After 6 months more bone was found in defects treated with PRP and radiographic examination showed enhanced maturity and bone consolidation of the PRP treated graft. A number of companies has recently commerzialized kits to prepare PC in autologous plasma. The level of platelets and growthfactors also level of fibrinogen might depend on the commercial kit being used.one important question to answer regarding the use of PRP is the optimal concentration of platelets. It is well known, that growth factors only stimulate bone healing beyond a certain threshold. Since most growth factors are found in bloodcells and not in the plasma, a correlation between level of bloodcells and bone healing is expected. However this has only been proved on TGF-β Since growth factor levels might be individually associated with sex, age, smoking etc., the growth factor levels should ideally be determined in each preparation to ensure activity. But since PRP preparation is often done at the same time as the operation takes place, platelet counts as a quality control is considered good manufacture practise By adding thrombin to the plasma, a gel is formed and platelets are activated 175. Theoretically not only platelets but also the gel might stimulate bone formation. Fibrin adhesives have been used in many surgical areas since early 70 s. It can be prepared from autologous 149, single donor or pooled plasma samples. Fibrinogen products such as Tissel (Immuno, Austria) are commercially available and is used for numerous applications to seal lungs, trachea, vascular anastomoses and liver- and kidney injuries. Fibrin sealant makes bone chips easier to handle 88,149, however the influence on bone healing is controversial 82. Bone might be formed on the surface of fibrin as reported from biopsies from impacted bone allograft 161. Side 14 af 40

15 Table V: Platelet concentrations in a number of platelet concentrate studies Preparation Animal PRP Platelet conc. Mean increase Reference (*10 9 /l) (%) No commercial kit Human Marx RE 88 No commercial kit Human Dugrillon, A 29 PCCS Human Weibrich, G 168,169 Curasan Human Weibrich, G 168,169 No commercial kit Canine Kim, ES 61 AGF Canine Jensen, TB 52 PCCS Canine Jensen, TB 54 No commercial kit Canine study IV and V Bone growth factors and grafted implants A large number of purified growth factors has previously been investigated in combination with bone graft and bone substitutes in bone defects. BMP-3 stimulates bone ingrowth in HA in baboons 116,117. BMP-2 alone increases ingrowth in HA in calvaria defects in rabbits 96,97 and OP-1 has previously increased ingrowth in HA in sheeps 27. bfgf stimulates bone ingrowth and increases vasular invasion into bone grafts and porous HA depending on dose 69,166. Addition of rhbmp-2 to autograft increases the expression of other BMPs and genes associated with bone formation and is capable of increasing bone formation in the central zone of spinal fusion masses in rabbits. In an unloaded bone chamber, the addition of rhbmp-2 increased the number of osteoclasts and amount of woven bone and fibrous tissue. Since it is well documented, that growth factors increase bone incorporation of bone graft and HA, growth factors might as well increase bone incorporation of grafted titanium implants. Soballe et al, found no effect of OP-1 on fixation of loaded implants in a primary setting. But in a revision with delayed bone healing properties, OP-1 increased fixation, however only at low concentrations 133. This indicates, that the greatest response of growth factors might be found in models and clinical situations with impaired healing capacity. Also OP-1 has been used in a clinical trial in combination with bone allograft in the revision of failed THA. However the trial was stopped due to some early failures 6. Table VI: Enhancement of bone allograftet implants using growth factors and platelet concentrates Side 15 af 40

16 Growth factor Carrier Graft Model Obs. period Animal Result Ref. rhop-1 No carrier fresh frozen loaded, non-ha coated, gap, 4 weeks canine no effect on fixation Søballe, K 133 bone allograft trabecular bone rhop-1 No carrier fresh frozen revision, loaded, non-ha coated, 4 weeks canine ffect at lower concentration Søballe, K 133 bone allograft gap, trabecular bone AGF No carrier fresh frozen unloaded, non-coated, gap, 3 weeks canine effect on bone ingrowth and fixation Jensen, TB 54 bone allograft trabecular bone PRP No carrier fresh frozen unloaded, HA coated, gap, 3 weeks canine no effect on bone ingrowth or implant Study IV bone allograft trabecular fixation PRP No carrier fresh frozen or unloaded, HA coated, gap, 3 weeks canine no effect on bone ingrowth or fixation StudyV processed bone trabecular bone allograft rhop-1 Collagen fresh frozen unloaded, HA coated, gap, 3 weeks canine effect on bone ingrowth Study I bone allograft trabecular rhop-1 Collagen ProOsteon unloaded, HA coated, gap, 3 weeks canine effect on bone ingrowth and implant Study I trabecular fixation rhop-1 Collagen ProOsteon/fresh unloaded, HA coated, gap, 3 weeks canine effect on bone ingrowth and implant Study II frozen bone trabecular fixation allograft rhop-1 Collagen Bone allograft loaded knee prostheses 6 weeks rabbit no effect Jeppsson, C 55 rhop-1 Collagen Fresh frozen unloaded, HA coated, gap, 6 weeks canine no effect Lind, M 73 bone allograft trabecular PCCS No carrier Allograft Non-loaded, non-ha coated, gap, trabecular bone 4 weeks canine effect on bone ongrowth and fixation Jensen, TB 52 Bone growth factors and non-grafted implants The ability of increasing bone incorporation of ungrafted implants has been investigated in a number of studies. TGF-β absorbed to TCP or HA coatings increases implant fixation and gap healing, however the effect is highest at lower concentrations 25,75,76,140. Cook et al found no effect of OP-1on bone ingrowth in dental implants inserted press fit after 12 weeks 23. OP-1 in a collagen carrier had little effect on implants inserted in trabecular bone surrounded by a gap. Table VI: Studies on enhancement of implant fixation using growth factors or PRP with no graft Growth factor Carrier Model Obs. period Animal Result Ref. Bovine OP* Collagen Interference fit after tooth ekstraction, non-ha coated 3 weeks Monkey Qualitative evaluation, increased ingrowth Ruther-ford, RB 121 rhop-1 Collagen Unloaded, +/- HA coating, gap, trabecular bone 6 weeks Canine Effect on fixation bone ingrowth and gap Lind, M 78 healing compared to empty gap but only on fixation compared to collagen rhop-1 Collagen Interference fit after tooth ekstraktion, +/- HA coating 12 Canine No effect Cook, SD 23 weeks TGF-β1 Implant Loadede +/- TCP coating, gap 6 weeks Canine Effect on ingrowth, no effect on fixation Lind, M 75 TGF-β1 Implant Unloaded +/- TCP coating, gap 6 weeks Canine Effect on fixation and bone ingrowth Lind, M 76 TGF-β1 Implant Unloaded, HA coatede, gap 6 weeks Canine Effect on fixation and bone TGF-β1 Lind, M 74 TGF-β1 Implant Unloadede, HA and TCP coatede, gap, 4 weeks Canine Effect on bone ingrowth and gap healing Sumner, RE 145 PRP No carrier Unloaded, HA coated, gap, trabecular bone 3 weeks Canine No effect Study IV and V AGF No carrier Loaded, non-ha coated, gap, trabecular bone 4 weeks Canine No effect Jensen, TB 52 *bovine OP consist ofbmp-2a and OP Side 16 af 40

17 Materials and methodological conciderations Ethical conciderations Canines bred for research were used in all studies. All animal handling was approved by Danish Control Board for Animal Research. Animals A long list of animals from mice to baboons are being used in orthopaedic research. We chose canines for several reason. Observation time, animal mobilisation and post operative care depend on species and we have more than 10 years of experience using canines allowing us to compare new data with previous results. Also canine bone have similar composition as human bone 1. The size of the animal is critical using the Soballe model and the use of smaller animals such as rabbits is not possible. Animals such as goats or sheeps have been used in implant research and might be used in our research group in future projects 3. We used same breed of dogs, at appr. same age and weight to decrease the biological variation. Observation time We used a relatively short observation period of three weeks for several reasons. Remodelling in canine bone has been estimated to be 2-3 times that of humans 64. We used young, but skelletal mature, healthy dogs and did not do any attempts to decrease bone healing. A gap of 2.5 mm surrounding a HA coated implant inserted in trabecular bone in a healthy canine heals after 6 weeks 139. Therefore all the gaps in our studies, grafted or non grafted, would probably heal after a time period and a short observation time was thus essential. A short observation time is interesting from a clinical point of view since early anchorage of the implant is an important predictor of long term survival 59. Implants We used porous coated titanium implants plasma sprayed with HA. Titanium porous surfaces are osteoconductive and the bioactivity is further enhanced by HA coating 102. As a consequence Soballe et al found strong effect of bone grafting in gaps surrounding non-hacoated implants but no effect of bone grafting when the implants were HA coated 139. Differences between treatment groups might easilier have been demonstrated using non-ha coated implants. HA coatings similar to the one we used in our experimental studies now show good clinical results 85,119. Experimental model Implants were surrounded by 3.0 mm gaps (study I and II and III) or 2.5 mm gaps (study IV and V). The implants were non-loaded and stable inserted extraarticularly in the femoral condyles 139 (study I, II and III) or the proximal humerus 105 (IV and V). This model was developed by Soballe and has been used for more than a decade in Orthopaedic Research Group to investigate the influence of various surface coatings and textures, growth factors, bone allografts, operation techniques and bone substitutes 53,74-78, ,107,112,113. The border of the drill hole was surrounded by trabecular bone in the femoral condyles whereas the border of the drill hole in some areas was in contact with cortical bone in the humerus. Primary cementless THA rely on cancellous bone ingrowth in the proximal femur. However in revisions Side 17 af 40

18 with extensive bone resorption, only a cortical shell might be left or the defect might even be uncontained. We used a non loaded model allowing no micromotions to occur. Implants in the clinical settings are loaded eventhough full weight loading is often avoided the first months postoperatively. Load plays a keyrole in incorporation and remodelling of bone allograft (Wollfs law). This model is thus less clinical relevant compared to other models previously been used in experimental orthopaedic research. The main reason for choosing the non-weight loaded model was, that it has less noise. We could have used a loaded Soballe model 103,104,107,112,113, However this model only allows a gap of 0.75 mm which might be too small to further increase bone grafted implants by adding growth factors. THA has previously been inserted in canines and goats This provide more clinical relevant data. However important parameters such as the volume of the graft, load and micromotions might not be easy to standardize in such model so the biological variation might increase significantly. Therefore more animals would be needed and only one grafting material would be investigated in each animal increasing the expenses and number of animals. Design The size and anatomy of the canines allowed insertion of implants in trabecular bone in each femoral condyle and two implants in each proximal humerus. Two studies were designed in each dog, each study using a paired design. Due to differences in loading and possible differences in mineral densities between humerus and femur, data humerus and femur should not be compared. Also, bone healing in the medial vs the lateral condyle in the femur and proximal vs distal humerus might differ due to differences in bone density. The different treatment groups were block randomized to the different locations. The paired design within each animal gives a higher statistical power since variation is decreased 68. Sample size The error of first kind (2α) was selected to be 5% and error of second error (β) to be 20%. Based on previous studies we estimated SD to be 50% of the mean. The minimal difference between treatment groups to be detected was set to be 70%. Based on these assumptions, the minimum number of animals was calculated to be seven 102. Eight dogs was thus included in each study. Grafting materials Fresh frozen bone allograft (study I-V) Proximal humerus, proximal femur and proximal tibia were harvested from two dogs in study I-II and III and one dog in study IV and V. After three weeks the bone graft was thawed and soft tissue and cartilage was removed. Using the finest grater in a standard bone mill, the graft was milled to chips which could be used in a 2.5 mm gap. These chips sizes are smaller than recommended in revision surgery. Smaller chips incorporate and resorb faster 108. Bacterial cultures ensured, that the graft was not contaminated. The graft was packed as tightly as possible. Impaction does decrease ingrowth, however tight impaction ensures implant stability. Side 18 af 40

19 Processed bone allograft (study IV) Freshly morsellized bone allograft was further processed by lavage, defattening in ethanol, freeze drying and irradiation. These steps are similar to recommendations by American Red Cross. ProOsteon (study I,II and III) ProOsteon 200 (Interpore,Irvine,US) is a corraline porous hydroxyapatite bone substitute approved by the FDA which has been on the market for more than two decades. The osteoconductive properties are well documented. Coral originally consists of 99% calcium carbonat 120 which is chemically transformed into almost non-resorbable HA 120. ProOsteon 200 was delivered as granules with a diameter of 425 µm to 1000 µm and a mean porous diameter of 230 µm and interconnection diameter of. Void fraction of ProOsteon 200 is 63%, mean trabecular thickness is ProOSteon was standardized by weight, put into containers and autoclaved according to manufactures instructions. OP-1 device (study I,II and III) OP-1 was used in study I, II and III. OP-1 is considered one of the most potent bone stimulating growth factors. Furthermore, it is commercially available for clinical use. Recent studies on OP-1 have not encouraged further use of OP-1 in revision surgery, however at the time of our experiment no studies on the effect of OP-1 on bone allograft was published. We could as well have used BMP-2 which was available through another company. OP-1 was delivered in a device consisting of 2.5 mg recombinant human OP-1 in 1 gram of bovine type I collagen (Stryker Biotech). Using this formula, we can not conclude whether the effect of OP-1 is due to OP-1 or the collagen carrier. Bovine collagen type I stimulates human osteoblasts in vitro and collagen enhances integration of bone substitutes in vivo. Collagen has osteoconductive properties with high bioactivity probably because it is capable of binding circulating factors such as osteonectin and growth factors. Surprisingly, collagen I alone proved to be just as efficient as collagen+op-1 to promote bone ongrowth to non-cemented HA coated and non-coated implants emphasizing the high bioactivity of collagen. The effect of OP-1 depends on concentration 20,21. The dosage of OP-1 in the present study was 300 µg OP-1 in 120 mg collagen carrier used in a 0.75 cc gap. Determination of concentration was based on studies by Cook in non-unions in canines 20,21. He concluded that concentrations beyond a certain threshold did not further increase bone formation. Platelet Rich Plasma (study IV and V) The preparation of PRP was done following the same procedure as described by Marx 88. Platelets and leucocytes counts in the PRP were appr. 770% and 910% compared to venous blood (table VIII). Also erythrocyt count was increased. Table VIII, analyses of PRP and whole blood. PRP/whole blood was calculated for every single dog. (median(range)) Group Baseline Count PRP PRP/whole blood Platelets (*10 9 /l) n=8 246 ( ) 1884 ( ) 7.7 ( ) Leukocytes(*10 9 /l) n=8 8.1 ( ) 71.7 ( ) 9.1 ( ) Erythrocytes(*10 12 /l) n=8 6.0 ( ) 8.6 ( ) 1.5 ( ) Side 19 af 40

20 Given our negative results in study IV and V, a test of the level of growth factors or a positive test on a cell culture would have been recommended. At the time of surgery, the commercial kits for preparation of PRP such as Symphony and AGF were not available. Mechanical evaluation The object of the mechanical test in study I, II, IV and V was to evaluate the bone-implant surface interface mechanically. The fixation of an implant is determined by the direct bonding of tissue, of which bone is believed to give the best fixation. Also bone interlock on a porous coated implant might play a role 102. We used a destructive push-out test which has been used in several studies. A pull-out or torque test could have been an alternative. However no mechanical test can mimic the clinical load, which is not only axial but also involves bending, shearing and compression. As an alternative to the destructive push-out test, we could have chosen a cyclic test 51. Thickness of implants varied from mm and push-out data was normalized by the surface area of the tested implant. Clearance (distance from the surface of the hole in the support jig to the surface of the implant) was set to 500 µm as suggested by Dhert 28. Ultimate shear strength, stiffness and energy absorption were determined on the load-displacement curves as previously described 102,134. Energy absorption has been suggested to be the most important mechanical parameter 102. A high stiffness might minimize micromotions, but a low energy absorption could lead to failure 102. The outcome of push-out test is affected by a number of parameters 102. We reduced the potential risks of variations due to storing, machine calibration, temperature, centralization over the support jig etc. by doing the tests in each study the same day, in random order and done blindly. The aim of the mechanical test in study III was to test the mechanical properties of incorporated bone graft and ProOsteon. This test was performed by centralizing the grafted 11 mm gap over a hole of 11.3 mm in diameter. A piston 10 mm in diameter applied load on the gap (paper III, figure 1 and 2). This test is a combination of compression of the bone loaded by the piston and the metal platform and a push-out test with stress applied to the interface at the border of the drillhole. The failure was always seen at the border of the drillhole. A similar test has previously been used to test newly formed bone in craniotomies 18. Side 20 af 40